A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidin e 5,6-double bond by oxidation, reduction or hydration. The biological outc ome includes increased mutation rate and potential lethality. A major DNA N -glycosylase responsible for the excision of modified pyrimidine bases is t he base excision repair (BER) glycosylase endonuclease Iii, for which funct ional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archa ea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyroba culum aerophilum, whose optimal growth temperature is 100 degreesC, The pre dicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclea se III (EcNth), The histidine-tagged recombinant protein was expressed in E .coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degre esC, PaNth displays DNA glycosylase/beta -lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT), This activity is enhanced when DH T is paired with G. Our data, showing the structural and functional similar ity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea, Conserved amino acid residues a re identified for five subfamilies of endonuclease III/UV endonuclease homo logs clustered by phylogenetic analysis.